The long term goal of the proposed research is to achieve a detailed understanding of the mechanism of nuclear pre-mRNA splicing (both cis- and trans-) using cell free extracts derived from embryos of the parasitic nematode, Ascaris. Trans-splicing is used as a mechanism of pre-mRNA processing in a variety of medically relevant parasites, including nematodes, trypanosomatid protaozoa and trematodes; the Ascaris system permits biochemical analysis of this reaction. Four Specific Aims are proposed: 1. Trans-spliceosome assembly involves independent recognition of the 3' splice site on the trans-splice acceptor and the 5' splice site on the SL RNA (the trans-splice donor). A combination of biochemical fractionation and site-specific crosslinking approaches will be used to characterize factors required for 3' splice site recognition. In addition, the hypothesis that the SL RNA is recruited to the acceptor as part of a pre-formed quadruple snRNA will be directly tested.; 2. Accurate maturation of nematode pre-mRNAs requires that the SL be excluded from internal (cis)- 3' splice acceptor sites. Evidence suggests that the cis- 5' splice site and the 5' splice site of the SL RNA are both present and may compete within the cis-spliceosome. Site-directed mutagenesis of both 5' splice sites will be used to determine how a particular 5' splice site is selected for the first transesterification reaction; 3. Tethered (site-specific) hydroxyl radical cleavage will be used to probe the RNA environments of the 5' splice site, the branch point, and the 3' splice site in staged cis- and trans-spliceosomes.; 4. Large (mg) quantities of staged spliceosomes will be purified for the purpose of biophysical analyses; electron cryomicroscopy and X-ray crystallography. In combination, the proposed biochemical and biophysical studies should provide new insight into the mechanism of trans-splicing in particular, and the mechanism of pre-mRNA splicing in general. Finally, a thorough understanding of trans-splicing may suggest possible avenues for therapeutic intervention in diseases caused by parasitic organisms that employ this pre-mRNA processing pathway.